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A kind of hypochlorous acid two-photon fluorescent probe hcp and its preparation method and application

A two-photon fluorescence, hypochlorous acid technology, applied in the field of biochemistry, can solve the problem of no fluorescent probe, and achieve the effect of inhibiting cell apoptosis and reducing oxidative stress

Active Publication Date: 2022-03-08
NANJING NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there have been no reports of direct real-time visualization of HOCl flux in the brain of living animals, especially during seizures.
Furthermore, no fluorescent probes are currently available for high-throughput screening of antiepileptic drugs to control MPO-mediated oxidative stress
To meet these needs, there are great challenges in the development of brain imaging agents, including efficient blood-brain barrier (BBB) ​​penetration, high specificity, good sensitivity, and desired photophysical properties

Method used

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  • A kind of hypochlorous acid two-photon fluorescent probe hcp and its preparation method and application
  • A kind of hypochlorous acid two-photon fluorescent probe hcp and its preparation method and application
  • A kind of hypochlorous acid two-photon fluorescent probe hcp and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0057] A kind of preparation method of the two-photon fluorescent probe HCP for hypochlorous acid, its preparation process is as follows:

[0058]

[0059] Compound 1: N,N-Dimethyl-p-phenylenediamine (5g, 36.7mmol, 1.0eq.) was dissolved in hydrochloric acid (6M, 66mL) and crotonaldehyde (6.0mL, 73.5mmol, 2eq.) was added, stirred at room temperature Afterwards, toluene (35 mL) was added to further reflux reaction overnight. The toluene was removed by cooling, and the aqueous layer was neutralized with saturated sodium hydroxide solution. Extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 4:1 v / v) to obtain compound 2: 6-(dimethylamino)quinoline-2-methyl.

[0060] Selenium dioxide (2.8g, 25.1mmol, 1.3 equiv.) was heated and stirred in dioxane / water (v / v=10 / 1), ...

Embodiment 2

[0064] Synthesis and Preliminary Evaluation of HCP

[0065] In vitro spectral response of HCP to HOCl. First detect the spectral properties of HCP in phosphate buffered saline (PBS) (10mM, pH 7.4, 5% DMSO), HCP (quantum yield Φ, 0.025) itself shows a maximum absorption peak at 451nm and a fluorescence peak at 581nm . To test the response of HCPs to HOCl, absorption and fluorescence spectroscopy were performed under physiological conditions. After adding 50μM HOCl, the absorption and fluorescence peaks of 10μM HCP were significantly reduced, while the characteristic blue-shifted absorption and fluorescence emission peaks appeared at 396nm and 495nm, respectively. ( figure 1 Middle A and B) Time-dependent fluorescence studies show that the enhancement of fluorescence intensity is within 5 seconds, and the reaction speed is very fast ( figure 1 Middle C), the fluorescence intensity is enhanced by 138 times after reacting with HOCl. High-performance liquid chromatography-mass...

Embodiment 3

[0067] HCP selectivity

[0068] The specificity of HCPs was investigated by analyzing the fluorescence response upon addition of various analytes. Such as figure 1 As shown in middle D, the selectivity of HCP for HOCl is superior to that of other biologically relevant molecules, such as other competing reactive oxygen or nitrogen species (ROS / RNS) ( OH, O 2 -, 1 o 2 , H 2 o 2 , OBr - , TBHP, HOCl, NO, NO 2 - , NO 3 - ), biothiols (H 2 S, GSH, Cys) and metal ions (Cu 2+ , Fe 3+ and Zn 2+ ). Only HOCl can promote a significant enhancement of 10 μM HCP emission at 495 nm in PBS solution at pH 7.4 containing 5% DMSO; HCP fluorescence was not observed even at higher concentrations (10 equiv) in the presence of other interfering substances Significant changes in the spectrum. In enhancing the fluorescence intensity at 495 nm, HCP is >40-fold and >80-fold more selective for HOCl than other species. It should be noted that hypobromous acid (HBrO, pKa 8.8) can be produ...

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Abstract

The invention discloses a hypochlorous acid two-photon fluorescent probe HCP and its preparation method and application. The two-photon fluorescent probe HCP has a structure as shown in structural formula I: the two-photon fluorescent probe HCP of the present invention can effectively The probe HCP can effectively and selectively image HOCl in vitro and in vivo, and can effectively cross the blood-brain barrier with targeting brain characteristics. The properties of the probe, the first in vivo and in vitro direct observation of upregulation of HOCl during KA-induced epilepsy, could be applied in the preparation of a tool for screening hypochlorous acid inhibitors. In addition, the present invention discloses for the first time that anti-epileptic inhibitors can be screened by combining high-content analysis with HCP, which will provide a simple and effective product and method for studying HOCl in biological systems and screening drugs for the prevention and treatment of epilepsy.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and relates to a hypochlorous acid two-photon fluorescent probe HCP and its preparation method and application, especially the design and synthesis of the two-photon fluorescent probe HCP for hypochlorous acid and its demonstration in epilepsy tracking imaging. Background technique [0002] Oxidative stress, mediated by myeloperoxidase (MPO), has been implicated in the pathological dysfunction of the epileptic brain. However, there are currently no powerful brain imaging tools for real-time detection of endogenous HOCl produced by MPO, and no fluorescent probes for rapid high-throughput screening of antiepileptic drugs to control MPO-mediated chloride stress. The dynamics of MPO activity and the pathological mechanisms of the endogenous HOCl flux mediated by it in seizures are still not fully understood. Myeloperoxidase (MPO) is a peroxidase containing two iron molecules that catalyzes the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07D215/38C07D471/04C09K11/06G01N21/64
CPCC07D215/38C07D471/04C09K11/06G01N21/6428G01N21/6486C09K2211/1029C09K2211/1074
Inventor 钱勇邵晨雯
Owner NANJING NORMAL UNIVERSITY
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